Spatially confined programmable DNA hydrogel scaffolds accelerate Cas12a trans-cleavage for digital bioimaging

Molecule diffusion limits target detection in conventional homogeneous solution, typically resulting in inefficient target-probe collision efficiency, low reaction kinetics, and insufficient signal accumulation. Herein, a dual rolling circle amplification-based DNA hydrogel was constructed to establish a hydrogel confined digital imaging platform (HCDIP) for visual imaging. In HCDIP, the spatial confinement provided by the DNA hydrogel scaffold constrains target recognition, signal conversion, and Cas12a cleavage in a confined three-dimensional network. Kinetic analysis showed that HCDIP activated Cas12a trans-cleavage activity rapidly, resulting in faster signal accumulation and earlier maximum reaction rate. Furthermore, fluorescence recovery after photobleaching and finite element simulation demonstrated that the HCDIP exhibited mass-transfer characteristics of restricted diffusion and local enrichment, with the effective diffusion coefficients of 45 nt and 150 nt nucleic acids reduced by approximately 2.8-fold and 3.3-fold, respectively, while maintaining a higher local concentration at the reaction center. By integrating HCDIP with an aptamer-based strategy, the visual detection of ochratoxin A (OTA) was achieved in a range of 2 pg·mL^-1-100 ng·mL^-1 with a detection limit of 1.5 pg·mL^-1. Owing to the programmability of the DNA hydrogel, HCDIP can be extended for orthogonal detection of multiple food contaminants, providing a general strategy for confined CRISPR-based ultrasensitive bioanalysis.